Principles of the Procedure Cylinder Plate Assay

Assay Media Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 8 Medium 9 Medium 10 Medium 11 Medium 12 Medium 13 Medium 19 Intended Use Assay Media are used for determinin antibiotic potency by the microbioloical assay technique. 1 3 These media, where noted, meet United States Pharmacopeia (USP) performance specifications. Summary and Explanation The activity (potency) of an antibiotic can be demonstrated under suitable conditions by its inhibitory effect on microoranisms. 2 Reduction in antimicrobial activity may reveal chanes not demonstrated by chemical methods. 2 assays are performed by the cylinder plate method and the turbidimetric tube assay. The cylinder plate method, first described by Abraham et al. 4 for the assay of penicillin, was later modified by Foster and Woodruff 5 and by Schmidt and Moyer. 6 assay media are prepared accordin to the specifications of the USP 2, European Pharmacopeia 7 and AOAC International. 3 The antibiotic media are identified numerically with names assined by Grove and Randall in Assay Methods of s. 1 Medium 19 corresponds to the use described in Outline of Details for Official Microbioloical Assays of s. 8 Medium 12 is prepared from the Grove and Randall formula. 1 They recommended its use for preparin test plates for the cylinder plate assay of the antifunal aents, nystatin and anisomycin, usin only a seed layer containin Saccharomyces cerevisiae as the test oranism. It is used for the assay of amphotericin B. Medium 1 and Medium 4 are used in a cylinder plate method for detectin penicillin in nonfat dry milk. 9 The use of standardized culture media and careful control of all test conditions are fundamental requisites in the microbioloical assay of antibiotics in order to achieve satisfactory test results. Principles of the Procedure Cylinder Plate Assay This method is based on the diffusion of an antibiotic solution from a cylinder placed on the surface of an inoculated aar medium. The diameter of a zone of inhibition after incubation depends, in part, on the concentration or activity of the antibiotic. This method is used in the assay of commercial preparations of antibiotics, as well as in the quantitative determination of antibiotics in body fluids, animal feeds and other materials. Turbidimetric Assay The turbidimetric method is based on the inhibition of rowth of a microbial culture in a fluid medium containin a uniform solution of an antibiotic. 2 Turbidimetric determinations have the advantae of requirin a short incubation period, providin test results after 3 or 4 hours. However, the presence of solvents or other inhibitory materials may influence turbidimetric assays more markedly than cylinder plate assays. Use of this method is appropriate only when test samples are clear. Formulae Medium 1 (Penassay Seed Aar) Beef Extract... 1.5 Yeast Extract... 3.0 Pancreatic Diest of Casein... 4.0 Peptone... 6.0 Dextrose... 1.0 Aar... 15.0 Medium 2 (Penassay Base Aar) Beef Extract... 1.5 Yeast Extract... 3.0 Peptone... 6.0 Aar... 15.0 Medium 3 (Penassay Broth) Beef Extract... 1.5 Yeast Extract... 1.5 Peptone... 5.0 Dextrose... 1.0 Sodium Chloride... 3.5 Dipotassium Phosphate... 3.68 Monopotassium Phosphate... 1.32 & BBL Manual, 2nd Edition

Also Known As DIFCO BRAND PRODUCT NAME ALTERNATive DIFCO NAME BBL BRAND PRODUCT NAME USP 2 AOAC 3 Medium 1 Penassay Seed Aar Medium 1 Aar Medium A Medium 2 Penassay Base Aar Medium 2 Aar Medium C Medium 3 Penassay Broth Assay Broth Medium 3 Broth Medium A Medium 4 Yeast Beef Aar Medium 4 Aar Medium B Medium 5 Streptomycin Assay Aar Medium 5 Aar Medium E Medium 8 Medium 8 Aar Medium D Medium 9 Polymyxin Base Aar Medium 9 Medium 10 Polymyxin Seed Aar Medium 10 Medium 11 Neomycin Assay Aar Medium 11 Aar Medium J Medium 12 Sabouraud Liquid Broth, Modified Medium 13 Broth Medium B Medium 19 Medium 19 User Quality Control Identity Specifications DEHYDRATED APPEARANCE SOLUTION PREPARED APPEARANCE PH AT 25 C Medium 1 Beie, homoeneous, free-flowin. 3.05% solution, soluble in purified water upon boilin. Solution is liht to medium amber, slihtly Liht to medium amber, slihtly 6.55 ± 0.05 Medium 2 Liht tan, homoeneous, free-flowin. 2.55% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht-medium amber, slihtly 6.55 ± 0.05 Medium 3 Tan, free-flowin, homoeneous. 1.75% solution, soluble in purified water upon boilin. Solution is liht to medium amber, clear. Liht to medium amber, clear. 7.0 ± 0.05 BBL Assay Broth ( Medium 3) Fine, homoeneous, free of extraneous material. 1.75% solution, soluble in purified water upon boilin. Solution is very pale to liht, yellow to tan, clear to slihtly hazy. Pale to liht, yellow to tan, clear to slihtly hazy. 7.0 ± 0.2 Medium 4 Liht tan, free-flowin, homoeneous. 2.65% solution, soluble in purified water upon boilin. Solution is liht amber, very slihtly Liht amber, very slihtly to slihtly 6.55 ± 0.05 Medium 5 Liht tan, free-flowin, homoeneous. 2.55% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht to medium amber, slihtly 7.9 ± 0.1 Medium 8 Liht tan, free-flowin, homoeneous. 2.55% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht to medium amber, slihtly 5.85 ± 0.05 Medium 9 Liht beie, free-flowin, homoeneous. 5.0% solution, soluble in purified water upon boilin. Solution is liht to medium amber, slihtly opalescent, may have a sliht flocculent precipitate. Liht to medium amber, slihtly opalescent with sliht flocculent precipitate. 7.25 ± 0.05 Medium 10 Beie, homoeneous, moist with a tendency to clump. 5.2% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht to medium amber, slihtly 7.25 ± 0.05 Medium 11 Beie, homoeneous, free-flowin. 3.05% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht to medium amber, slihtly 7.95 ± 0.05 Medium 12 Tan, homoeneous, free-flowin. 6.25% solution, soluble in purified water upon boilin. Solution is liht to medium amber, very slihtly to slihtly Liht to medium amber, slihtly 6.1 ± 0.1 BBL Sabouraud Liquid Broth, Modified ( Medium 13) Fine, homoeneous, free of extraneous material. 3.0% solution, soluble in purified water upon boilin. Solution is liht to medium, yellow to tan, clear to slihtly hazy. Liht to medium, yellow to tan, clear to slihtly hazy. 5.7 ± 0.1 Medium 19 Liht tan, homoeneous, free-flowin. 6.0% solution, soluble in purified water upon boilin. Solution is medium amber, very slihtly to slihtly Medium amber, slihtly 6.1 ± 0.1 & BBL Manual, 2nd Edition

Cultural Response Medium 1 Medium 2 method and incubate at 35 ± 2 C for 18-24 hours. Oranism ATCC Inoculum CFU RECOVERY Staphylococcus aureus 6538P 30-300 Good Medium 3 Prepare the medium per label directions. Inoculate and incubate at 35 ± 2 C for up to 24 hours. Oranism ATCC Inoculum CFU RECOVERY Enterococcus faecium 10541 ~10 7 Good Escherichia coli 10536 ~10 7 Good Klebsiella pneumoniae 10031 ~10 7 Good Staphylococcus aureus 6538P ~10 7 Good BBL Assay Broth ( Medium 3) Prepare the medium per label directions. Inoculate and incubate at 25 ± 2 C for the Saccharomyces cerevisiae and 35 ± 2 C for the remainin oranisms for 7 days. Oranism ATCC Inoculum CFU recovery Bacillus subtilis 6633 10 3 Good Escherichia coli 10536 10 3 Good Kocuria rhizophila 9341 10 3 Good Saccharomyces cerevisiae 9763 10 3 Good Staphylococcus aureus 6538P 10 3 Good Medium 4 method and incubate at 35 ± 2 C for 40-48 hours. Oranism ATCC Inoculum CFU RECOVERY Kocuria rhizophila 9341 30-300 Good Medium 9 Medium 10 method and incubate at 35 ± 2 C for 40-48 hours. Oranism ATCC Inoculum CFU RECOVERY Bordetella bronchiseptica 4617 30-300 Good Medium 11 method and incubate at 35 ± 2 C for 18-48 hours. Oranism ATCC Inoculum CFU RECOVERY Kocuria rhizophila 9341 30-300 Good Staphylococcus epidermidis 12228 30-300 Good Medium 12 Medium 19 method and incubate at 30 ± 2 C for 40-48 hours. Oranism ATCC Inoculum CFU RECOVERY Saccharomyces cerevisiae 2601 30-300 Good BBL Sabouraud Liquid Broth, Modified ( Medium 13) Prepare the medium per label directions. Inoculate and incubate at 25 ± 2 C for 7 days (use one loopful of a fresh 3-7 day culture for A. brasiliensis and T. mentarophytes). Oranism ATCC Inoculum CFU recovery Asperillus brasiliensis (nier) 16404 Undiluted Good Candida albicans 60193 10 3 Good Saccharomyces cerevisiae 9763 10 3 Good Trichophyton mentarophytes 9533 Undiluted Good Medium 5 Medium 8 method and incubate at 35 ± 2 C for 18-24 hours. Oranism ATCC Inoculum CFU RECOVERY Bacillus subtilis 6633 30-300 Good BBL Assay Broth ( Medium 3) Beef Extract... 1.5 Yeast Extract... 1.5 Pancreatic Diest of Gelatin... 5.0 Dextrose... 1.0 Sodium Chloride... 3.5 Dipotassium Phosphate... 3.68 Monopotassium Phosphate... 1.32 Medium 4 (Yeast Beef Aar) Beef Extract... 1.5 Yeast Extract... 3.0 Peptone... 6.0 Dextrose... 1.0 Aar... 15.0 Medium 5 (Streptomycin Assay Aar).Same as Medium 2, except for the final ph after autoclavin. Medium 8.Same as Medium 2, except for the final ph after autoclavin. Medium 9 (Polymyxin Base Aar) Pancreatic Diest of Casein... 17.0 Soy Peptone... 3.0 Dextrose... 2.5 Sodium Chloride... 5.0 Dipotassium Phosphate... 2.5 Aar... 20.0 Medium 10 (Polymyxin Seed Aar) Pancreatic Diest of Casein... 17.0 Soybean Peptone... 3.0 Dextrose... 2.5 Sodium Chloride... 5.0 Dipotassium Phosphate... 2.5 Aar... 12.0 Polysorbate 80... 10.0 Medium 11 (Neomycin Assay Aar).Same as Medium 1, except for the final ph after autoclavin. & BBL Manual, 2nd Edition

Selection of Media for the Microbioloical Assay of s 2 Cylinder Plate Cylinder Plate Inoculum Base Layer Seed Layer Turbidimetric Assay Method Oranism ATCC Medium Medium medium assay medium Amikacin Turbidimetric Staphylococcus aureus 29737 1 3 Amphotericin B Cylinder Plate Saccharomyces cerevisiae 9763 19 19 Bacitracin Cylinder Plate Micrococcus luteus 10240 1 2 1 Candicidin Turbidimetric Saccharomyces cerevisiae 9763 19 2 13 Capreomycin Turbidimetric Klebsiella pneumoniae 10031 1 3 Carbenicillin Cylinder Plate Pseudomonas aeruinosa 25619 1 9 10 Cephalothin Cylinder Plate Staphylococcus aureus 29737 1 2 1 Cephapirin Cylinder Plate Staphylococcus aureus 29737 1 2 1 Chloramphenicol Turbidimetric Escherichia coli 10536 1 3 Chlortetracycline Turbidimetric Staphylococcus aureus 29737 1 3 Cloxacillin Cylinder Plate Staphylococcus aureus 29737 1 2 1 Colistimethate, sodium Cylinder Plate Bordetella bronchiseptica 4617 1 9 10 Colistin Cylinder Plate Bordetella bronchiseptica 4617 1 9 10 Cycloserine Turbidimetric Staphylococcus aureus 29737 1 3 Demeclyocycline Turbidimetric Staphylococcus aureus 29737 1 3 Dihydrostreptomycin Cylinder Plate Bacillus subtilis 6633 32* 5 5 Dihydrostreptomycin Turbidimetric Klebsiella pneumoniae 10031 1 3 Doxycycline Turbidimetric Staphylococcus aureus 29737 1 3 Erythromycin Cylinder Plate Kocuria rhizophila 9341 1 11 11 Gentamicin Cylinder Plate Staphylococcus epidermidis 12228 1 11 11 Gramicidin Turbidimetric Enterococcus hirae 10541 3 3 Kanamycin Turbidimetric Staphylococcus aureus 29737 1 3 Methacycline Turbidimetric Staphylococcus aureus 29737 1 3 Nafcillin Cylinder Plate Staphylococcus aureus 29737 1 2 1 Neomycin Cylinder Plate Staphylococcus epidermidis 12228 1 11 11 Neomycin Turbidimetric Klebsiella pneumoniae 10031 1 39** Netilmicin Cylinder Plate Staphylococcus epidermidis 12228 1 11 11 Novobiocin Cylinder Plate Staphylococcus epidermidis 12228 1 2 1 Nystatin Cylinder Plate Saccharomyces cerevisiae 2601 19 19 Oxytetracycline Turbidimetric Staphylococcus aureus 29737 1 3 Paromomycin Cylinder Plate Staphylococcus epidermidis 12228 1 11 11 Penicillin G Cylinder Plate Staphylococcus aureus 29737 1 2 1 Polymyxin B Cylinder Plate Bordetella bronchiseptica 4617 1 9 10 Rolitetracycline Turbidimetric Staphylococcus aureus 29737 1 3 Sisomicin Cylinder Plate Staphylococcus epidermidis 12228 1 11 11 Streptomycin Turbidimetric Klebsiella pneumoniae 10031 1 3 Tetracycline Turbidimetric Staphylococcus aureus 29737 1 3 Tobramycin Turbidimetric Staphylococcus aureus 29737 1 3 Troleandomycin Turbidimetric Klebsiella pneumoniae 10031 1 3 Tylosin Turbidimetric Staphylococcus aureus 9144 3 39** Vancomycin Cylinder Plate Bacillus subtilis 6633 32* 8 8 * Same as Medium 1, except for the additional inredient of 300 m of mananese sulfate. ** Same as Medium 3, except that the final ph after autoclavin is 7.9 ± 0.1. Medium 12 Beef Extract... 2.5 Yeast Extract... 5.0 Peptone... 10.0 Dextrose... 10.0 Sodium Chloride... 10.0 Aar... 25.0 BBL Sabouraud Liquid Broth, Modified ( Medium 13) Pancreatic Diest of Casein... 5.0 Peptic Diest of Animal Tissue... 5.0 Dextrose... 20.0 & BBL Manual, 2nd Edition

Medium 19 Beef Extract... 2.4 Yeast Extract... 4.7 Peptone... 9.4 Dextrose... 10.0 Sodium Chloride... 10.0 Aar... 23.5 *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product 1. Suspend the powder in 1 L of purified water: Medium 1 30.5 ; Medium 2 25.5 ; Medium 3 17.5 ; BBL Assay Broth ( Medium 3) 17.5 ; Medium 4 26.5 ; Medium 5 25.5 ; Medium 8 25.5 ; Medium 9 50 ; Medium 10 52 ; Medium 11 30.5 ; Medium 12 62.5 ; BBL Sabourand Liquid Broth, Modified ( Medium 13) 30 ; Medium 19 60. Mix thorouhly. 2. Heat with frequent aitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121 C for 15 minutes. 4. To raise the ph of Medium 11 to 8.3 ± 0.1, cool the base to 45-50 C and add NaOH. 5. Test samples of the finished product for performance usin stable, typical control cultures. Procedure Test Oranism Preparation Maintain stock cultures on aar slants and make transfers at 1- or 2-week intervals. Prepare the inoculum for assay by washin rowth from a fresh 24-48 hour aar slant usin sterile purified water, saline or Medium 3 and further dilute the culture to obtain the desired oranism concentration. In some turbidimetric assays, an 18- to 24-hour culture of the test oranism in Medium 3, diluted to obtain the optimal number of oranisms, is used. When Bacillus subtilis is used as the test oranism, inoculate it on Medium 1 and incubate at 37 C for 1 week, wash spores from the aar surface, and heat the spores at 56 C for 30 minutes. Wash the spores three times in purified water, heat aain at 65 C for 30 minutes, and then dilute to the optimal concentration. This inoculum preparation should produce a sharp zone in the assay. Medium 1 modified by the addition of 300 m mananese sulfate (MnSO 4 H 2 O) per liter often aids the sporulation of B. subtilis and may be used in preparin the spore suspension. When B. cereus var. mycoides is required, inoculate the oranism on Medium 1 and incubate at 30 C for 1 week. Wash and prepare the spores as for B. subtilis, above. Cylinder Plate Assay Use 20 100 mm lass or plastic Petri dishes with sufficient depth so that cylinders used in the assay will not be pushed into the medium by the cover. Use stainless steel or porcelain assay cylinders havin the followin dimensions (± 0.1 mm): 8 mm outside diameter, 6 mm inside diameter and 10 mm lon. 2 Carefully clean the cylinders to remove all residues, usin an occasional acid bath (i.e., with approximately 2N nitric acid or with chromic acid). 2 Four or six cylinders are enerally used per plate, evenly spaced on a 2.8 cm radius. To assure accurate assays, work on a level surface to obtain uniformly thick base and seed layers in the Petri dish. Allow the base layer to solidify and then overlay the seed layer containin a proper concentration of the test oranism. The amount of medium in the layers varies for different antibiotics, with most assays specifyin a 21 ml base layer and a 4 ml seed layer. In any case, dishes with flat bottoms are required to assure complete coverae of the bottom of the dish when small amounts of base medium are used. Tilt the plate to obtain even coverae of the base layer by the seed layer and allow it to solidify in a level position. Plates should be used the same day as prepared. Turbidimetric Assay Use lass or plastic test tubes (i.e., 16 125 mm or 18 150 mm) that are relatively uniform in lenth, diameter and thickness and substantially free from surface blemishes. 2 Tubes that will be placed in the spectrophotometer should be matched and free of scratches or blemishes. 2 Clean the tubes thorouhly to remove all antibiotic residues and traces of cleanin solution and, prior to subsequent use, sterilize tubes that have been previously used. 2 Prepare workin dilutions of the antibiotic reference standards in specific concentrations. To a 1 ml quantity of each solution in a suitable tube, add 9 ml of inoculated broth, as required. Prepare similar solutions of the assay materials containin approximately the same amounts of antibiotic activity and place in tubes. Incubate the tubes for 3-4 hours at the required temperature, enerally in a water bath. At the end of the incubation period, stop rowth by addin 0.5 ml of 1:3 formalin. Determine the amount of rowth by measurin liht transmittance with a suitable spectrophotometer. Determine the concentration of the antibiotic by comparin the rowth obtained with that iven by reference standard solutions. For a complete discussion of antibiotic assay methods, refer to appropriate procedures outlined in the references. 2,3,7 & BBL Manual, 2nd Edition

Expected Results Refer to appropriate procedures for results. 2,3,7 References 1. Grove and Randall. 1955. Assay methods of antibiotics. Medical Encyclopedia, Inc. New York, N.Y. 2. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md. 3. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC International, Gaithersbur, Md. 4. Abraham, Chain, Fletcher, Florey, Gardner, Heatley and Jennins. 1941. Lancett ii:177. 5. Foster and Woodruff. 1943. J. Bacteriol. 46:187. 6. Schmidt and Moyer. 1944. J. Bacteriol. 47:199. 7. Council of Europe. 2002. European pharmacopeia, 4th ed. Council of Europe, Strasbourh, France. 8. Kirshbaum and Arret. 1967. J. Pharm. Sci. 56:512. 9. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed. American Public Health Association, Washinton, D.C. Availability Medium 1 AOAC EP USP Cat. No. 226340 Dehydrated 500 Medium 2 AOAC USP Cat. No. 227020 Dehydrated 500 Medium 3 AOAC EP USP Cat. No. 224320 Dehydrated 500 224310 Dehydrated 2 k BBL Assay Broth ( Medium 3) AOAC EP USP Cat. No. 210932 Dehydrated 500 Medium 4 Cat. No. 224410 Dehydrated 500 Medium 5 Cat. No. 227710 Dehydrated 500 Medium 8 Cat. No. 266710 Dehydrated 500 Medium 9 EP USP Cat. No. 246210 Dehydrated 500 Medium 10 EP USP Cat. No. 246310 Dehydrated 500 * Medium 11 Cat. No. 259310 Dehydrated 500 Medium 12 Cat. No. 266910 Dehydrated 500 BBL Sabouraud Liquid Broth Modified ( Medium 13) AOAC USP Cat. No. 210986 Dehydrated 500 221014 Prepared Tubes (K Tubes) Pk. of 10 Medium 19 EP USP Cat. No. 243100 Dehydrated 500 Europe Cat. No. 254655 Prepared Bottles, 250 ml Pk. of 10 *Store at 2-8 C. & BBL Manual, 2nd Edition